A wide swath of bacterial pathogens grow within deep tissue sites during disease. Pathogen growth in these sites results in the recruitment of immune cells that attempt to clear of the invader, but these cells are often ineffective because the virulent organism blocks the clearing process. As a consequence, the microorganism sets up a beachhead where it can either establish a persistent infection or venture to spread throughout the host. Depending on the nature of the recruited cells and tissue damage that occurs, these foci of infection are referred to as abscesses, microabscesses, granulomas, or some combination of processes. For many pathogens that grow outside of host cells, distinct microcolonies are formed, predicted to result in considerable intermicrobial communication and direct targeting of host cells surrounding the colony. An overriding problem in the infectious disease field is that the resulting architecture can only be established in animal infection models and cannot be maintained or analyzed in culture. This work proposes to overcome this stumbling block. Yersinia pseudotuberculosis is an enteropathogenic bacterium that can spread from the intestine into regional lymph nodes, the liver and the spleen, establishing microcolonies surrounded by layers of neutrophils, macrophages and inflammatory monocytes. The bacterium directly inactivates nearby neutrophils, but there is a compensating attack by distal macrophages that generates nitric oxide (NO) and its antimicrobial derivatives. Bacteria on the periphery of the microcolony inactivate NO, protecting their centrally localized kin from exposure to toxic metabolites. The proposed Research Plan will exploit a bioengineered gel microdroplet system to accurately reconstruct this battle. The Aims propose to analyze Y. pseudotuberculosis interaction with immune cells, by growing bacteria in microcolonies within the gel droplets, surrounding the droplets with adherent activated macrophages, morphologically mimicking a true infectious site. Using a fluorescent reporter readout, the transcriptional profiles of peripheral and centrally located bacteria will be analyzed, and compared to bacteria growing either in the absence of macrophage stress or in a nonstructured environment. The system will be used to identify bacterial transcriptional circuits that allow peripheral bacteria to maintain viability, and which protect the centrally located kin from attack. It will also identify the bacterial transcriptional response to growth in aggregates found in tissues, as well as identify previously uncharacterized physiological and stress responses of the small bacterial community to secreted macrophage products. Successful completion of the Aims is part of the long-term goal of determining how inter-bacterial interactions ensure the establishment of an infectious niche, and to evaluate how anti-microbial immune cells collaborate with pathogens to promote disease.